Our objectives are to understand the functions and organization of the human adenovius (Ad) early genes. We propose a coordinated investigation of early genes: early proteins (EP), early RNA transcription and processing, Ad DNA replication, and integration of Ad early genes in transformed cells and their expression as nuclear RNA and mRNA. Ad2 EPs will be purified and mapped on the viral genome by partial amino acid sequence and cell-free translation. These studies together with 5'-terminal sequencing of the 13 Ad2 mRNA T1 oligonucleotides and longer sequences mapped in early regions E1-E5 should provide insights into DNA sequences important in transcription initiation, RNA processing, splicing, and translation initiation. Functions of EPs will be studied using in vitro DNA replication and possibly RNA transcription systems, monospecific EP-antisera, enzyme assays, and analysis of the interaction of EPs (and cell proteins) with interesting Ad2 DNA fragments using protection experiments coupled with Maxam-Gilbert sequencing. Transcription studies by Northern blotting and nuclease gel procedures will emphasize E5 and transformed cell transcripts. HE C19 cells, an Ad12 transformed cell line that appears to have an intact late region will be used to investigate regulation. Ad integration will be studied in 5 group C transformed cell lines; Southern blotting studies are completed. We will clone 20 kb pieces of transformed cell DNA and study (1) possible cell and viral sequence specificity at the junction, and (2) possible cell DNA sequences required for Ad integration. Ad DNA replication studies will concentrate on the terminal protein (TP) and the reconstitution of in vitro Ad DNA replication with purified EPs, emphasizing initiation. TP studies will focus on (1) the protein-DNA linkage, (2) genetic origin (antisera, cell free translation, amino acid sequences of group A-E TPs, synthetic oligodeoxynuleotide probes coding TP amino acid sequences), and (3) interaction with Ad DNA, 73K DNA binding protein, and VA RNA, and (4) possible role in DNA replication. Studies on early gene organization and function will be important in illuminating cellular gene transcription and regulation, DNA replication, and growth control.